1. Field of the Invention
The present invention relates to a novel protein binding specifically to the Fc fragment of human immunoglobulin G(IgG), a gene coding for the said protein and to a process for producing the said protein.
2. Description of Related Art
It has been known that certain microorganisms produce a series of proteins known as bacterial Fc receptors which have affinity to the Fc fragment of immunoglobulin (Boyle et al., Bio/Technology 5, 697(1987)).
Typical examples of such proteins are Protein A derived from Staphylococcus aureus and Protein G derived from Streptococcus G148.
These Protein characteristically bind to the Fc fragment of immunoglobulin and are used for assay, purification and preparation of antibodies as well as clinical diagnosis and biological research.
They can also be used for treatment of cancers and autoimmune diseases in which the proteins immobilized onto insoluble carriers are used to adsorb or remove undesirable immune complexes from blood(Cancer 46, 675(1980)).
These known proteins have some undesirable properties as agents for purification of human monoclonal antibodies produced from non-human animal cells or for removal of excessive IgG from blood for the purposes of blood purification.
Protein A binds to IgGs of various animal species including human beings as well as human IgA, IgM and so on. Protein G binds only to IgG but it binds both to human IgG and to IgGs of other animal species (Fahnestock, Trends in Biotechnology 5, 79 (1987)). Thus, their binding specificities are not so narrow that they can be used for assay, purification and adsorption or removal of human IgG.
Under the circumstances, development of a protein capable of binding specifically to human IgG has been demanded.
It was suspected that a protein which binds to human IgG(IgG1, IgG2, IgG3 and IgG4) but not to IgGs of other animal species would be present in cells of group A Streptococcus strains (Bjorck, J. Immunol., 133, 969(1984)). However, no such protein has been isolated. Two types of IgG-Fc-binding proteins have been isolated from group A Streptococcus, one of which binds to human IgG(IgG1, IgG2 and IgG4), pig IgG and rabbit IgG; and the other binds specifically to human IgG3 (Boyle et al., Bio/Technology 5, 697(1987)).
It has been unknown whether or not such IgG-Fc-binding protein which binds specifically to human IgG (IgG1, IgG2, IgG3 and IgG4) but which does not bind to IgGs of most other animal species and to human IgA, IgD, IgE and IgM exists and whether or not a sufficient amount of such protein can stably be obtained.
The present inventors have extensively studied seeking for proteins which specifically binds to human IgG, and, as a result, found that Streptococcus sp. AP1 belonging to group A Streptococcus produces the protein with the above-mentioned properties. They have also obtained the gene coding for the said protein and provided a process for producing the said protein by the utilization of the gene through further extensive studies.
Thus, the present invention provides a novel protein capable of specifically binding to human IgG, and useful for assay and purification of human IgG, removal or adsorption of excessive IgG from blood and for diagnosis of autoimmune diseases. It also provide a gene coding for the said protein and a process for producing the said protcin in industrial scales.
The protein provided by the present invention, which is hereinafter referred to as Protein H, is a protein capable of binding to the Fc fragment of immunoglobulin and produced by a strain of group A Streptococcus and has the following binding specificity:
i) It binds to human IgG(IgG1, IgG2, IgG3 and IgG4) and rabbit IgG; PA0 ii) It does no& bind to IgGs of mouse, rat, bovine, sheep and goat; PA0 iii) It does not bind to human IgA, IgD, IgE and IgM; PA0 i) It strongly binds to human IgG(IgG1, IgG2, IgG3 and IgG4), human IgGFc and rabbit IgG; PA0 ii) It weakly binds to pig IgG; PA0 iii) It does not bind to IgGs of mouse, rat, bovine, sheep, goat and horse; PA0 iv) It does not bind to human IgGFab, IgA, IgD, IgE and IgM. PA0 i) inserting a gene coding for the Protein H into a vector; PA0 ii) introducing the resulting vector into a suitable host cell; PA0 iii) culturing the resulting transformant cell to produce the Protein H; and PA0 iv) recovering the Protein H from the culture.
or the following binding specificity:
The strain, Streptococcus sp. A which produces the Protein H has been deposited at the Fermentation Research Institute, Japan, under the deposit No. FERM P-10374 and also under the deposit No. FERM BP-2371 according to the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE.
The Protein H can be produced by the genetic engineering technology using the gene coding for the Protein H.
The Protein H produced from the gene isolated from the Streptococcus sp. APl has been found. to be of the amino acid sequence given in the FIG. 1(A-B) by the analysis of the DNA sequences of the gene.
It is to be understood that subfragments o variants of the Protein H specifically disclosed in the present application wherein original amino acid sequence is modified or changed by insertion, addition, substitution, inversion or deletion of one or more amino acids are within the scope of the present invention as far as they retain the essential binding specificity as mentioned above.
The gene coding for the Protein H can be isolated from the chromosomal DNA of a Protein H-producing stain such as Streptococcus sp. APl based on the information on the DNA sequence of the Protein H shown in FIG. 4(A-B). The isolation of the gene can also be carried out as follows:
The chromosomal DNA can be isolated from cells of the Protein H-producing strain in accordance with a known method (Fahnestock, J. Bacteriol. 167, 870(1986)). The isolated chromosomal DNA is then segmented into fragments of adequate lengths by biochemical means such as digestion with restriction enzyme or physical means.
The resulting fragments are then inserted at an adequate restriction site into an adequate cloning vector such as .lambda.gtll (Young et al., Proc. Natl. Acad. Sci. USA 80, 1194 (1983)) or plasmid vectors such as pUC18 (Messing et al., Gene 33, 103 (1985)).
The vectors are then incorporated into adequate host cells such as E. coli.
From the resulting transformants, the clones producing the protein which binds to human IgG or the Fc region of human IgG are selected by a known method (Fahnestock et al., J. Bacteriol., 167, 870 (1986)).
After the proteins capable of binding to human IgG or the Fc region of human IgG are isolated from the resulting positive clones according to a conventional method, the binding specificities of the proteins are determined to select the clones producing the Protein H. FIG. 2 shows the binding specificity of the Protein H.
After the DNA insert of the said clone is isolated by a conventional method, the DNA sequence of the insert is determined by a known method (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977); Choen et al., DNA 4, 165 (1985)). FIG. 4(A-B) shows the DNA sequence of the DNA insert isolated from the positive clone Fc4. FIG. 5(A-B) shows the DNA sequence of the structural gene coding for the Protein H isolated from the said clone.
It is necessary for genes to be expressed that they contain expression-controlling regions such as promoter, terminator and the like. The gene shown in FIG. 4 contains such as necessary expression-controlling regions.
The expression of genes may be effected with expression vectors having the necessary expression controlling regions in which only the structural gene is inserted. For this purpose, the structural gene shown in FIG. 5(A-B) can preferably be used. The structural gene coding for the Protein H can be obtained from the gene of FIG. 4(A-B) or synthesized by a conventional method based on the amino acid sequence given in the present specification.
As for the expression vectors, various host-vector systems have already been developed, from which the most suitable host-vector system can be selected for the expression of the gene of the present invention.
It has been known that, for each host cell, there is a particularly preferable codon usage for the expression of a given gene. In constructing a gene to be used for a given host-vector system, the codons preferable for the host should be used. Adequate sequences for the gene for the Protein H to be used in a particular host-vector system can be designed based on the amino acid sequence given in FIG. 1(A-B) and synthesized by a conventional synthetic method.
The present invention is further concerned with a process for producing the Protein H by culturing a host cell transformed with an expression vector in which the gene encoding the Protein H is inserted.
The process comprises steps of
In the first step, the gene coding for the Protein H, which is isolated from the chromosomal DNA of Streptococcus sp. API or synthesized as mentioned above, is inserted into a vector suitable for a host to be used for the expression of the Protein H. The insertion of the gene can be carried out by digesting the vector with a suitable restriction enzyme and linking thereto the gene by a conventional method.
In the second step, the resulting vector with the gene is introduced into host cells. The host cells may be Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae and the like. The introduction of the expression vector into the host cells can be effected in a conventional way.
In the third step, the resulting transformant cells are cultured in a suitable medium to produce the Protein H by the expression of the gene. The cultivation can be conducted in a conventional manner.
In the fourth step, the produced Protein H is recovered from the culture and purified, which can be conducted by a known method. For example, the cells are destroyed by a known method such as ultrasonification, enzyme treatment or grinding. The Protein H released out of the cells or secreted into the medium is recovered and purified by conventional methods usually used in the field of biochemistry such as ion-exchange chromatography, gel filtration, affinity chromatography using IgG as ligand, hydrophobic chromatography and reversed phase chromatography, which may be used solely or in suitable combinations.
As mentioned above, the protein provided by the present invention can be used for identification or separation of human IgG. For these purposes, the protein may be brought into a reagent kit or a pharmaceutical composition by mixing or combining it with suitable reagents, additives or carries.
The present invention will more precisely be described by the following examples. But, they are not intended to limit the scope of the present invention.